A growing number of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, and Creutzfeldt-Jakob disease (CJD), involve the misfolding of normal proteins in the brain, which has recently been associated with the binding of metal ions such as iron, copper, and zinc. It is thought that metal dyshomeostasis is involved in protein misfolding and leads to oxidative damage and neuron degeneration. Yet, the functions of these metal ions and the misfolded proteins in the disease process are not well understood. To date, metal concentrations in brain tissue are generally measured via macroscopic (bulk) techniques, which cannot provide any spatial information on localized metal accumulation. Stains and/or fluorescently-tagged antibodies are used to identify misfolded proteins in tissue, but do not provide direct information on the protein's structure. Structural studies involving metal-binding and protein misfolding are primarily done in vitro. Thus, this proposal aims to bridge the gap between macroscopic methods of analyzing brain tissue and in vitro studies of metal-protein binding. It describes the development and utilization of a combination of spectroscopic imaging tools that directly investigate metal content and protein structure within intact tissues. The overall goal of this proposal is to obtain an in situ structural and mechanistic picture of how metal ions in the brain are involved in protein misfolding and aggregation in two protein-folding diseases: Alzheimer's disease and scrapie. We hypothesize that elevated concentrations of metal ions (notably Cu, Fe, and Zn) in the brain are involved in the disease pathogenesis. Using x-ray microspectroscopy, we will image the metal ion distribution, concentration, oxidation state, and local structure as a function of disease severity in intact brain tissue. By combining fluorescence microscopy and infrared microspectroscopy, we will image the location and secondary structure of the associated misfolded proteins. By combining these results, we will identify which metal ions accumulate before, concurrently, or after protein misfolding. We will test whether metal ion reduction is correlated with oxidation markers such as lipid peroxidation in the brain. Finally, we use the local structure and homogeneity of the in situ metal-protein complex to develop a possible mechanism for the complex formation and toxicity.